Proteins play an important role in today's medical portfolio. For human application every pharmaceutical substance has to meet distinct criteria. To ensure the safety of biopharmaceutical agents to humans nucleic acids, viruses, and host cell proteins, which would cause severe harm, have to be removed especially. To meet the regulatory specification one or more purification steps have to follow the manufacturing process. Among other purity, throughput, and yield play an important role in determining an appropriate purification process.
Different methods are well established and widespread used for protein purification, such as affinity chromatography with thiophilic ligands, Cu-chelate, or microbial proteins (e.g., protein A or protein G affinity chromatography), ion exchange chromatography (e.g., cation exchange, anion exchange, and mixed-mode exchange), thiophilic adsorption, hydrophobic interaction or aromatic adsorption chromatography, size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).
Recombinant polypeptides can be produced e.g., by prokaryotic cells such as E. coli. The recombinantly produced polypeptide accounts for the majority of the prokaryotic cell's polypeptide content and is often deposited as insoluble aggregate, i.e., as a so called inclusion body, within the prokaryotic cell. For the isolation of the recombinant polypeptide the cells have to be disintegrated and the recombinant polypeptide contained in the inclusion bodies has to be solubilized after the separation of the inclusion bodies from the cell debris. For the solubilization chaotropic reagents, such as urea or guanidinium hydrochloride, are used. To cleave disulfide bonds reducing agents, especially under alkaline conditions, such as dithioerythriol, dithiothreitol, or β-mercaptoethanol are added. After the solubilization of the aggregated polypeptide the globular structure of the recombinant polypeptide, which is essential for the biological activity, has to be reestablished. During this so called renaturation process the concentration of the denaturating agents is slowly reduced, e.g., by dialysis against a suited buffer, which allows the denatured polypeptide to refold into its biologically active structure. After the renaturation is the recombinant polypeptide purified to a purity acceptable for the intended use. For example, for the use as a therapeutic protein a purity of more than 90% has to be established. Recombinantly produced polypeptides obtained from E. coli are normally accompanied by nucleic acids, endotoxins, polypeptides from the producing cell, and not-renaturated recombinant polypeptides.
With the number of different chromatographic methods available a multitude of combinations has to be tested in order to find a suitable purification process. In these combinations different sequences and even different numbers of chromatographic methods may be used. Thus, a method for determining a suitable sequence of chromatographic steps for the purification of a non-glycosylated polypeptide is desirable.
In WO 2007/075283 a multi step system and methods of target molecule purification are reported. Methods for purifying compounds comprising a protein of interest are reported in WO 2007/016250. A process for purifying a recombinant protein including one or a few procedural steps only is reported in WO 2006/101441. Rege et al. (Rege, K., Biotechnol. Bioeng. 93 (2006) 618-630) report a high-throughput process development for recombinant protein purification. In KR 2002/080108 a process for purifying human growth hormone from recombinant E. coli is reported.